PROJECT SUMMARY/ABSTRACT Tightly regulated flux of materials and signals across the exquisitely organized plasma membrane (PM) is essential for healthy cellular function. Disruption of this careful choreography is a common mechanism underlying the pathogenesis of many genetic and infectious diseases. Therefore, a central problem in cell biology is to understand the key molecular components that direct this intricate organization of signaling, transport and structural machinery at the PM. A phospholipid located in the cytosolic leaflet, phosphatidylinositol 4,5-bisphosphate (PIP2), is a key regulator of PM function, controlling recruitment and/or activation of this protein machinery. Yet how PIP2 levels are regulated to ensure each PM function has access to enough lipid to ensure correct operation, and how PIP2 is able to regulate each function discretely, is poorly understood. The goal of our research is therefore to develop a detailed mechanistic understanding of how cells regulate PIP2 levels in the PM, and how this facilitates regulation of individual PIP2-dependent functions. The goal of this application is to identify fundamental mechanisms in cell culture models, and to apply the new insights and approaches to physiological and disease-relevant systems through our established network of collaborators. Firstly, we will determine the nanoscopic organization of PIP2 molecules in the PM and determine their enrichment at sites of specific PM function. To accomplish this goal, we will probe and manipulate lipid enrichment at sites of cytoskeletal, signaling or trafficking functions with nanometer resolution, using super-resolution optical imaging approaches and chemical genetics. Secondly, we will delineate the mechanisms that regulate global PM PIP2 levels, by identifying the molecular components driving negative feedback of PIP2 synthesis. Thirdly, we will identify the biological functions of PIP2 5-phosphatase enzymes, as well as the mechanism of pathogenesis for disease-associated mutations in these enzymes. To accomplish this goal, we will identify where endogenous 5-phosphatase enzymes act in the cell, where PIP2 accumulates after loss of these enzymes, and what cellular phenotypes are triggered by the resulting accumulation of PIP2. We will employ innovative approaches throughout, combining super-resolution imaging of PIP2 and its myriad effector proteins with chemical genetics to acutely manipulate PIP2 with exquisite spatial and temporal precision. The proposed research is significant because it will uncover fundamental mechanisms that choreograph the interplay of PM functions, and consequently provide a crucial first step in developing new approaches to experimentally or therapeutically manipulate these functions in isolation.